Certain yeasts are able to utilize methanol as a sole source of carbon and energy. Species of the so-called methylotrophic yeasts that have the biochemical pathways necessary for methanol utilization are classified into four genera, based upon cell morphology and growth characteristics: Hansenula, Pichia, Candida, and Torulopsis (Billon-Grand, Mycotaxon 35:201 (1989); Kurtzman, Mycologia 84:72 (1992)). Not all species within these genera are capable of utilizing methanol as a source of carbon and energy, and therefore, individual species of a genus may differ in physiology and metabolism.
Methylotrophic yeasts are attractive candidates for use in recombinant protein production systems. Some methylotrophic yeasts have been shown to grow rapidly to high biomass on minimal defined media. Certain genes of methylotrophic yeasts are tightly regulated and highly expressed under induced or de-repressed conditions, suggesting that promoters of these genes might be useful for producing polypeptides of commercial value. See, for example, Romanos et al., Yeast 8:423 (1992), Cregg et al., Bio/Technology 11:905 (1993), Faber et al., Yeast 11:1331 (1995), and Jong et al., SIM News 46:199 (1996).
Development of methylotrophic yeasts as hosts for use in recombinant production systems has been slow, due in part to a lack of efficient promoters, selectable markers, and mutant host cells, as well as suitable transformation techniques. The most highly developed methylotrophic host systems utilize Pichia pastoris (Komagataella pastoris) and Hansenula polymorpha (Pichia angusta) (Faber et al, Curr. Genet. 25:305-310 (1994); Cregg et al., ibid.; Romanos et al., Yeast 8:423 (1992); U.S. Pat. No. 4,855,242; U.S. Pat. No. 4,857,467; U.S. Pat. No. 4,879,231; and U.S. Pat. No. 4,929,555).
For example, numerous fermentation processes have been described for expression of heterologous proteins by Pichia pastorsis. Typically, these methods are based on a fermentation recipe developed by Brieley et al., international publication No. WO 90/03431, which uses an initial growth phase on glycerol, followed by a period of glycerol feeding to build up the biomass (see, for example, Stratton et al., "High Cell-Density Fermentation," in Methods in Molecular Biology, Vol. 103, Higgins and Cregg (eds.), pages 107-120 (Humana Press Inc. 1998)). To induce protein expression using the methanol-inducible alcohol oxidase 1 (AOX1) promoter, a slow feed of methanol is initiated along with glycerol feeding. After the cells have adapted to growth on methanol, the glycerol feed is stopped, and methanol is used as the sole carbon source for the remainder of the fermentation. The basal recipe includes a very high level of inorganic salts, including magnesium sulfate, potassium sulfate, calcium sulfate, phosphoric acid, and trace metals. The combination of these salts forms an insoluble precipitate that easily falls out of solution.
A new methylotrophic yeast species, designated Pichia methanolica, has recently been developed for a heterologous expression system (Raymond et al., Yeast 14:11 (1998)). The use of the expensive carbon source glycerol for Pichia methanolica is not practical due to the yeast's poor growth on this substrate. Moreover, the high level of inorganic salts, which precipitate from the medium developed for Pichia pastoris, indicated that a new fermentation recipe would be beneficial for growth and protein expression by Pichia methanolica.
Raymond et al., Yeast 14:11 (1998), described a new recipe for P. methanolica that uses a filtered sterilized solution of phosphate glass (sodium hexametaphosphate) for a phosphate source. The benefits of using phosphate glass were described for E. coli fermentation where it does not form precipitates with other inorganic salts and glucose (see, for example, (Giusez et al., Protein Express. Purif 12:249 (1998)). One of the main drawbacks in using phosphate glass is that this component must be added separately as a filter-sterilized solution to the fermentor, and the basal medium may form a precipitate before addition of the phosphate glass. The other major drawback is that phosphate glass can inhibit the growth of P. methanolica.
Accordingly, there remains a need in the art for techniques that will facilitate the large-scale culture of methylotrophic yeasts, including Pichia methanolica, to produce polypeptides of economic importance.